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Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the United States, but the actual roles that eosinophils play in CRSwNP remain largely unclear., Objective: To reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue, we performed single cell RNA sequencing (scRNA-Seq) analysis of NP tissue., Methods: Sinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched before processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by Gene Ontology (geneontology.org) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry., Results: After filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses), and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were downregulated and 354 genes upregulated in NP eosinophils compared to all PB eosinophils (>1.5-fold, P adj  < .05). Upregulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling, and antiapoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue., Conclusions: Elevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part by controlling inflammation and hyperproliferation of other cells., Competing Interests: Disclosure statement Supported in part by Regeneron Pharmaceuticals, the National Institutes of Health (grants P01AI145818, R01AI137174, and U19AI136443), and a grant from the Ernest S. Bazley Foundation. Disclosure of potential conflict of interest: W. W. Stevens has served on advisory boards for GlaxoSmithKline, Regeneron, and Melinta Therapeutics. A. T. Petershas served on advisory boards for Sanofi-Genzyme/Regeneron, Optinose, AstraZeneca, Novartis, and GSK; and has received research support from Optinose and Sanofi/Regeneron. L. C. Grammer reports personal fees from Astellas Pharmaceuticals. K. C. Welch reports consultant fees from Baxter, OptiNose, and Acclarent. D. B. Conley reports consulting fees from Medtronic and Sanofi/Regeneron. R. P. Schleimer reports personal fees from Intersect ENT, Merck, GlaxoSmithKline, Sanofi, AstraZeneca/Medimmune, Genentech, Actobio Therapeutics, Lyra Therapeutics, Astellas Pharma, and Otsuka; and has royalty rights to Siglec-8 and Siglec-8 ligand–related patents licensed by Johns Hopkins to Allakos. R. C. Kern reports consulting fees from Lyra Therapeutics, Medtronic, GSK, Genentech and Sanofi/Regeneron. B. S. Bochner has received remuneration for serving on scientific advisory board of Allakos and owns stock in Allakos; has served as consultant for GSK, Third Harmonic Bio, Lupagen, Acelyrin, and Sanofi/Regeneron; receives publication-related royalty payments from Elsevier and UpToDate; is coinventor of existing Siglec-8–related patents and thus may be entitled to a share of royalties received by Johns Hopkins University during development and potential sales of such products; and is a cofounder of Allakos, which makes him subject to certain restrictions under university policy (terms of this arrangement are being managed by Johns Hopkins University and Northwestern University in accordance with their conflict-of-interest policies). B. K. Tan reports personal fees from Sanofi/Regeneron/Genzyme and GSK. A. Kato has served on advisory board for AstraZeneca; reports gift for his research from Lyra Therapeutics; and has received research grants from Regeneron and AstraZeneca. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Oncostatin M (OSM) may promote type 2 inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) by inducing thymic stromal lymphopoietin (TSLP)., Objective: We sought to study the impact of OSM on TSLP synthesis and release from nasal epithelial cells (NECs)., Methods: OSM receptors, IL-4 receptors (IL-4R), and TSLP were evaluated in mucosal tissue and primary NECs from patients with CRSwNP by quantitative PCR and immunofluorescence. Air-liquid interface-cultured NECs were stimulated with cytokines, including OSM, and quantitative PCR, ELISA, Western blot, and flow cytometry were used to assess the expression of OSM receptors, IL-4R, and TSLP., Results: Increased levels of OSM receptor β chain (OSMRβ), IL-4Rα, and TSLP were observed in nasal polyp tissues and primary epithelial cells from nasal polyps of patients with CRSwNP compared with control tissues or cells from control subjects. The level of expression of OSMRβ in tissue was correlated with levels of both IL-4Rα and TSLP. OSM stimulation of NECs increased the expression of OSMRβ and IL-4Rα. Stimulation with IL-4 plus OSM augmented the production of TSLP; the response was suppressed by a signal transducer and activator of transcription 6 inhibitor. Stimulation of NECs with IL-4 plus OSM increased the expression of proprotein convertase subtilisin/kexin 3, an enzyme that truncates and activates TSLP., Conclusions: OSM increases the expression of IL-4Rα and synergizes with IL-4 to induce the synthesis and release of TSLP in NECs. Because the combination of IL-4 and OSM also augmented the expression of proprotein convertase subtilisin/kexin 3, these results suggest that OSM can induce both synthesis and posttranslational processing/activation of TSLP, promoting type 2 inflammation., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Patients with aspirin-exacerbated respiratory disease (AERD) regularly exhibit severe nasal polyposis. Studies suggest that chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by excessive fibrin deposition associated with a profound decrease in epithelial tissue plasminogen activator (tPA). Retinoids, including vitamin A and its active metabolite retinoic acid (RA), are necessary for maintaining epithelial function and well-known inducers of tPA in endothelial cells., Objectives: This study sought to determine whether endogenous retinoids are involved in NP pathophysiology and disease severity in patients with CRSwNP and AERD., Methods: NP tissue was collected from patients with AERD or CRSwNP, and concentrations of retinoids and fibrinolysis markers were measured using ELISA. Normal human bronchial epithelial cells were stimulated alone or in combination with RA and IL-13 for 24 hours., Results: This study observed lower retinoid levels in nasal polyps of patients with AERD than those with CRSwNP or healthy controls (P &lt; .01). Levels of the fibrin-breakdown product d-dimer were the lowest in AERD polyps (P &lt; .01), which is consistent with lower tPA expression (P &lt; .01). In vitro, all-trans RA upregulated tPA levels in normal human bronchial epithelial cells by 15-fold and reversed the IL-13-induced attenuation of tPA expression in cultured cells (P &lt; .01)., Conclusions: RA, a potent inducer of epithelial tPA in vitro, is reduced in tissue from patients with AERD, a finding that may potentially contribute to decreased levels of tPA and fibrinolysis in AERD. RA can induce tPA in epithelial cells and can reverse IL-13-induced tPA suppression in vitro, suggesting the potential utility of RA in treating patients with CRSwNP and/or AERD., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps is frequently managed with endoscopic sinus surgery (ESS). Prior studies describe individual clinical variables and eosinophil density measures as prognostic for polyp recurrence (PR). However, the relative prognostic significance of these have not been extensively investigated., Objectives: We sought to evaluate the impact of PR on measures of disease severity post-ESS and quantify the prognostic value of various clinical variables and biomarkers., Methods: Ninety-four patients with chronic rhinosinusitis with nasal polyps and prospectively biobanked polyp homogenates at the time of ESS were recruited 2 to 5 years post-ESS. Patients were evaluated with patient-reported outcome measures and endoscopic and radiographic scoring pre- and post-ESS. Biomarkers in polyp homogenates were measured with ELISA and Luminex. Relaxed least absolute shrinkage and selection operator regression optimized predictive clinical, biomarker, and combined models. Model performance was assessed using receiver-operating characteristic curve and random forest analysis., Results: PR was found in 39.4% of patients, despite significant improvements in modified Lund-Mackay (MLM) radiographic and 22-item Sinonasal Outcomes Test scores (both P < .0001). PR was significantly associated with worse post-ESS MLM, modified Lund-Kennedy, and 22-item Sinonasal Outcomes Test scores. Relaxed least absolute shrinkage and selection operator identified 2 clinical predictors (area under the curve = 0.79) and 3 biomarkers (area under the curve = 0.78) that were prognostic for PR. When combined, the model incorporating these pre-ESS factors: MLM, asthma, eosinophil cationic protein, anti-double-stranded DNA IgG, and IL-5 improved PR predictive accuracy to area under the curve of 0.89. Random forest analysis identified and validated each of the 5 variables as the strongest predictors of PR., Conclusions: PR had strong associations with patient-reported outcome measures, endoscopic and radiographic severity. A combined model comprised of eosinophil cationic protein, IL-5, pre-ESS MLM, asthma, and anti-double-stranded DNA IgG could accurately predict PR., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Increased activation of the coagulation cascade and diminished fibrinolysis combine to promote fibrin deposition and polyp formation in chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP). More information is needed concerning mechanisms of coagulation in CRSwNP., Objective: We investigated the mechanisms as well as the initiation and regulation of coagulation cascade activation in CRS., Methods: Samples were collected from 135 subjects with CRSwNP, 80 subjects with chronic CRS without nasal polyps (NP), and 65 control subjects. The levels of activated factor X (FXa), prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex, tissue factor (TF), and TF pathway inhibitor (TFPI) were monitored in CRS by real-time PCR, ELISA, immunohistochemistry, or immunofluorescence. Heteromeric complexes of TF with activated factor VII (FVII) and TF with activated FVII and FXa were assessed by coimmunoprecipitation and Western blotting., Results: Increased levels of FXa, F1+2, and thrombin-antithrombin complex were detected in NP tissue compared to uncinate tissue from CRS and control subjects. Although free TF protein levels were not increased in NP, immunoprecipitation of TF in NP tissue revealed increased complexes of TF with FVII. Local expression of FVII was detected in sinonasal mucosa, and the ratio of TFPI to FXa was lower in NP tissue., Conclusion: The coagulation cascade is associated with NP compared to control and uncinate tissue from CRS patients, and TF and FVII are produced locally in sinonasal mucosa in patients. TF and FVII can activate the extrinsic coagulation pathway, suggesting that this pathway may activate fibrin deposition in CRSwNP. Reduced formation of the complex of FXa and TFPI in NP may reduce natural suppression of the extrinsic coagulation pathway in CRSwNP., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammatory disease of the upper airways. AZD1981 is a selective antagonist of chemoattractant receptor-homologous molecule expressed on T helper type 2 and other type 2 cells, including innate lymphoid cells type 2, eosinophils, and basophils., Objective: To evaluate the efficacy of AZD1981 in reducing nasal polyp size when added to intranasal corticosteroids in adult patients with CRSwNP., Methods: Eighty-one subjects (18-70 years of age) with CRSwNP were recruited and screened for trial eligibility from allergy and otolaryngology clinics from a single tertiary care site between June 2016 and August 2019. Eligible patients were randomized in a double-blind fashion to receive either AZD1981 (n = 22) or placebo (n = 21) orally three times a day for 12 weeks, added to intranasal corticosteroids. The primary endpoint was a change in nasal polyp score (NPS) at 12 weeks. Secondary endpoints included improvement in sinus computed tomography using Lund Mackay scoring, symptoms using visual analog scale, quality of life using Sino Nasal Outcome Test-22, and the Brief Smell Identification Test., Results: Forty-three patients met the inclusion criteria and were enrolled. At 12 weeks, there was no difference in NPS change in the AZD1981 arm (mean 0, standard error 0.34, n = 15) compared with placebo (mean 0.20, standard error 0.36, n = 17); mean difference -0.20 (95% confidence interval: -1.21, 0.81; p = .69). No significant differences were observed for Lund Mackay score, symptoms, quality of life, or smell test. AZD1981 was well tolerated except for one case of hypersensitivity reaction., Conclusion: In patients with CRSwNP, the addition of AZD1981 to intranasal corticosteroids did not change nasal polyp size, radiographic scores, symptoms, or disease-specific quality of life., (© 2022 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Background: Chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is well characterized by type 2 (T2) inflammation characterized by eosinophilia in Western countries. However, the presence and roles of neutrophils in T2 CRSwNP are poorly understood., Objective: We sought to clarify accumulation and inflammatory roles of neutrophils in CRSwNP in a Western population., Methods: Sinonasal tissues and nasal lavage fluids were obtained from control patients and patients with CRS, and neutrophil markers were determined by ELISA. The presence of neutrophils in tissue was determined by flow cytometry. The gene expression profiles in neutrophils were determined by RNA sequencing., Results: A neutrophil marker elastase was selectively elevated in nasal polyp (NP) tissue, whereas eosinophilic cationic protein (an eosinophil marker) was elevated in both uncinate and NP tissues of CRSwNP patients. Nasal lavage fluid myeloperoxidase (another neutrophil marker) was also significantly elevated in CRSwNP compared to control patients. Neutrophil markers were more greatly elevated in CRSwNP patients with recurrent disease. Flow cytometric analysis confirmed that neutrophil numbers were significantly elevated in NPs compared to control tissues. RNA sequencing analysis found that 344 genes were >3-fold and significantly elevated in NP neutrophils compared to peripheral blood neutrophils. Gene Ontology analysis suggested that the elevated genes in NP neutrophils were significantly associated with activation. Results suggest that neutrophils are accumulated in T2 NP tissues and that accumulated neutrophils are highly activated and contribute to inflammation in NPs., Conclusions: Neutrophils may play a heretofore unrecognized meaningful role in the pathogenesis of CRSwNP in Western countries and may be a potentially important therapeutic target in T2 CRSwNP., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Aspirin-exacerbated respiratory disease (AERD) is characterized by the triad of chronic rhinosinusitis with nasal polyps (CRSwNP), asthma, and intolerance to cyclooxygenase-1 enzyme inhibitors. The underlying mechanisms contributing to AERD pathogenesis are not fully understood, but AERD is characterized by an enhanced type 2 inflammatory phenotype. Basophils are potent type 2 effector cells, but their involvement in AERD pathophysiology remains unclear., Objective: We sought to characterize the systemic and local basophil responses in patients with AERD compared with patients with CRSwNP., Methods: Sinonasal tissues including inferior turbinate and/or nasal polyps (NPs) and peripheral blood were collected from controls, patients with AERD, and patients with CRSwNP. Expression of cell surface (CD45, FcεRI, CD203c), activation (CD63), and intracellular (2D7) markers associated with basophils was characterized using flow cytometry. Clinical data including Lund-Mackay scores and pulmonary function were obtained., Results: The mean number of basophils (CD45 + CD203c + FcεRI + CD117 - ) detected in AERD NPs (147 ± 28 cells/mg tissue) was significantly elevated compared with that detected in CRSwNP NPs (69 ± 20 cells/mg tissue; P = .01). The number of circulating basophils was significantly elevated in patients with AERD (P = .04). Basophils in NPs had significantly higher CD203c and CD63 mean fluorescence intensity compared with blood in both conditions (P < .01). Basophils from AERD NPs had lower expression of the granule content marker 2D7 compared with those from matched blood (P < .01) or NPs of patients with CRSwNP (P = .06), suggesting ongoing degranulation. Basophil 2D7 mean fluorescence intensity significantly correlated with pulmonary function (r = 0.62; P = .02) and inversely correlated with sinonasal inflammation (r = -0.56; P = .004)., Conclusions: Increased basophil numbers and extent of ongoing degranulation in NPs of patients with AERD compared with patients with CRSwNP may contribute to the exaggerated disease pathogenesis and severity unique to AERD., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 10 11  pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10 ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5 days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease subdivided based on the presence or absence of nasal polyps (NPs). Histologic features of chronic rhinosinusitis with nasal polyps (CRSwNP) include inflammatory cell infiltration and excessive fibrin deposition in NPs. Thrombin-activatable fibrinolysis inhibitor (TAFI) is an enzyme that plays an antifibrinolytic role in the body. The significance of TAFI has been documented in patients with chronic inflammatory diseases, including chronic lung disease; however, it has not been evaluated in the pathogenesis of NPs., Objective: The objective of this study was to evaluate the potential role of TAFI in the pathogenesis of NPs., Methods: Nasal lavage fluid was collected from control subjects and patients with CRS. We measured levels of thrombin/anti-thrombin complex (TATc) and TAFI protein using an ELISA., Results: TATc levels in nasal lavage fluid were significantly increased in patients with CRSwNP and patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with control subjects, and TAFI levels in nasal lavage fluid were also significantly increased in patients with CRSwNP compared with those in control subjects and patients with CRSsNP. There was a significant correlation between TATc and TAFI levels in nasal lavage fluid. Interestingly, patients with CRS and asthma showed increased TATc and TAFI levels in nasal lavage fluid compared with those in patients with CRS without asthma, especially patients with CRSwNP., Conclusions: Increased TATc and TAFI levels in nasal passages of patients with CRSwNP might participate in fibrin deposition in NPs and might play a role in the pathogenesis of CRSwNP and asthma., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease generally divided based on the presence or absence of nasal polyps (NPs). One of the features of NPs is excessive fibrin deposition, which is associated with down-regulation of tissue plasminogen activator (t-PA) in NPs. As t-PA is expressed in epithelial cells, and epithelium is readily accessible to topical therapies, identifying compounds that can mediate the induction of t-PA would be a potential new strategy for the treatment of NPs., Objective: The objective of this study was to determine whether short-chain fatty acids (SCFAs) can induce t-PA in airway epithelial cells via their known receptors GPR41 and GPR43., Methods: We performed immunohistochemistry (IHC) to determine whether receptors for SCFAs, known as G protein-coupled receptor 41/free fatty acid receptor 3 (GPR41/FFAR3) and GPR43/FFAR2, are expressed in nasal tissue. Primary normal human bronchial epithelial (NHBE) cells were stimulated with different concentrations of SCFAs to test induction of t-PA, which was analysed by expression of mRNA and protein. Mediation of responses by SCFA receptors was evaluated by specific receptor gene silencing with siRNA., Results: Immunohistochemistry study revealed that airway epithelial cells expressed GPR41 and GPR43. Acetic acid, propionic acid, butyric acid and valeric acid significantly induced t-PA expression from two- to tenfolds. The strongest inducer of t-PA from NHBE cells was propionic acid; cells stimulated with propionic acid released t-PA into the supernatant in its active form. Gene silencing of GPR41 and GPR43 revealed that induction of t-PA by SCFAs was dependent upon both GPR41 and GPR43., Conclusions and Clinical Relevance: Short-chain fatty acids were shown to induce airway epithelial cell expression of t-PA via GPR41 and GPR43. Topical delivery of potent compounds that activate these receptors may have value by reducing fibrin deposition and shrinking nasal polyp growth., (© 2018 John Wiley & Sons Ltd.)

Background: IgD is an enigmatic antibody isotype best known when coexpressed with IgM on naive B cells. However, increased soluble IgD (sIgD) levels and increased IgD + IgM - B-cell populations have been described in the human upper respiratory mucosa., Objective: We assessed whether levels of sIgD and IgD + B cell counts are altered in nasal tissue from patients with chronic rhinosinusitis (CRS). We further characterized IgD + B-cell populations and explored clinical and local inflammatory factors associated with tissue sIgD levels., Methods: sIgD levels were measured by means of ELISA in nasal tissues, nasal lavage fluid, sera, and supernatants of dissociated nasal tissues. IgD + cells were identified by using immunofluorescence and flow cytometry. Inflammatory mediator levels in tissues were assessed by using real-time PCR and multiplex immunoassays. Bacterial cultures from the middle meatus were performed. Underlying medical history and medicine use were obtained from medical records., Results: sIgD levels and numbers of IgD + cells were significantly increased in uncinate tissue (UT) of patients with chronic rhinosinusitis without nasal polyps (CRSsNP) compared with that of control subjects (4-fold, P < .05). IgD + cells were densely scattered in the periglandular regions of UT from patients with CRSsNP. We also found that IgD + CD19 + CD38 bright plasmablast numbers were significantly increased in tissues from patients with CRSsNP compared with control tissues (P < .05). Among numerous factors tested, IL-2 levels were increased in UT from patients with CRSsNP and were positively correlated with tissue IgD levels. Additionally, supernatants of IL-2-stimulated dissociated tissue from patients with CRSsNP had significantly increased sIgD levels compared with those in IL-2-stimulated dissociated control tissue ex vivo (P < .05). Tissue from patients with CRS with preoperative antibiotic use or those with pathogenic bacteria showed higher IgD levels compared with tissue from patients without these variables (P < .05)., Conclusion: sIgD levels and IgD + CD19 + CD38 bright plasmablast counts were increased in nasal tissue of patients with CRSsNP. IgD levels were associated with increased IL-2 levels and the presence of pathogenic bacteria. These findings suggest that IgD might contribute to enhancement mucosal immunity or inflammation or respond to bacterial infections in patients with CRS, especially CRSsNP., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) has a high propensity for recurrence. Studies suggest that eosinophilia influences disease severity and surgical outcomes, but the selection of sinonasal site for measuring eosinophilia has not been examined. The aim of this study was to investigate how region-specific tissue eosinophilia affects radiographic severity, comorbidity prevalence, and polyp recurrence risk following sinus surgery., Methods: Eosinophil cationic protein (ECP) levels in uncinate tissue (UT) and nasal polyp (NP) homogenates from 116 CRSwNP patients were measured using enzyme-linked immunosorbent assay (ELISA). Clinical history, radiographic severity, and time to polyp recurrence were obtained from electronic health records. The correlations between baseline Lund-Mackay scores and comorbidities were compared between UT and NP ECP levels. Cox regression and Kaplan-Meier analysis were then performed to assess whether UT or NP ECP better predicted recurrence. Censoring occurred at 4 years or at last follow-up if there was no endoscopic diagnosis of recurrent polyps., Results: Lund-Mackay scores were significantly correlated with UT and NP ECP (r = 0.46 and 0.26 respectively, p < 0.05). UT but not NP ECP was significantly higher in patients with asthma (p < 0.01) and aspirin-exacerbated respiratory disease (AERD) (p < 0.05). Polyp recurrence risk was only significantly higher for patients with eosinophilic UT tissue (hazard ratio [HR] = 2.84, p = 0.025). When measured in NP, eosinophilia did not predict recurrence., Conclusion: Although ECP in NP was higher than in UT tissue, eosinophilia in UT tissue was a more clinically coherent biomarker of baseline radiographic severity, comorbid asthma and AERD, and prospective polyp recurrence risk than NP eosinophilia., (© 2017 ARS-AAOA, LLC.)

Background: Microparticles (MPs) are submicron-sized shed membrane vesicles released from activated or injured cells and are detectable by flow cytometry. MP levels have been used as biomarkers to evaluate cell injury or activation in patients with pathological conditions., Objective: We sought to compare MP types and levels in nasal lavage fluids (NLFs) from controls and patients with chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD)., Methods: We collected NLFs from patients with CRSsNP (n = 33), CRSwNP (n = 45), and AERD (n = 31) and control (n = 24) subjects. Standardized flow cytometry methods were used to characterize the following MP types: endothelial MPs, epithelial MPs (epithelial cell adhesion molecule [EpCAM](+)MPs, E-cadherin(+)MPs), platelet MPs (CD31(+)CD41(+)MPs), eosinophil MPs (EGF-like module-containing mucin-like hormone receptor-like 1[EMR1](+)MPs), mast cell MPs (high-affinity IgE receptor [FcεRI](+)c-kit(+)MPs), and basophil MPs (CD203c(+)c-kit(-)MPs). Basophil activation was evaluated by the mean fluorescence intensity of CD203c on basophil MPs., Results: Activated mast cell MPs (CD137(+) FcεRI(+)c-kit(+)MPs) were significantly increased in NLFs of controls compared with NLFs of patients with CRSsNP (2.3-fold; P < .02), CRSwNP (2.3-fold; P < .03), and AERD (7.4-fold; P < .0001). Platelet MPs (3.5-fold; P < .01) and basophil MPs (2.5-fold; P < .05) were increased only in patients with AERD. Mean fluorescence intensity of CD203c on MPs was increased in patients with CRSwNP (P < .002) and AERD (P < .0001), but not in patients with CRSsNP. EpCAM(+)MPs in patients with CRSwNP were no different from control (P = .91) and lower than those in patients with CRSsNP (P < .02) and AERD (P < .002)., Conclusions: Based on released MPs, mast cells, platelets, and basophils were more highly activated in patients with AERD than in patients with CRS. Epithelial injury was lower in patients with CRSwNP than in patients with CRSsNP and AERD. MP analysis may help identify phenotypes of CRS, and in distinguishing AERD from CRSwNP., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease of the nose and paranasal sinuses that presents without or with nasal polyps (CRSwNP). Notable features of CRSwNP are the frequent presence of type 2 allergic inflammation and high prevalence of Staphylococcus aureus (SA) colonization. As inflammation persists, sinus tissue undergoes epithelial damage and repair along with polyp growth, despite active medical management. Because one feature of damaged tissue is enhancement of growth factor signaling, we evaluated the presence of epidermal growth factor receptor (EGFR) ligands and matrix metalloproteinases (MMPs) in CRS. The objectives of this study were to analyze the expression of EGFR ligands and MMPs in patients with CRS and to investigate the possible role of SA on epithelial activation. Sinonasal tissues were collected during surgery from control subjects and patients with CRS. Tissues were processed as described previously for analysis of mRNA (RT-PCR) and proteins (ELISA) for the majority of EGFR ligands within the tissue extracts. CRS tissue was used for evaluation of the distribution of epiregulin (EREG), an EGFR ligand, and MMP-1 by immunohistochemistry. In parallel studies, expression of these genes and proteins was analyzed in cultured primary airway epithelial cells. Elevated expression of EREG and MMP-1 mRNA and protein was observed in uncinate and polyp tissue from patients with CRSwNP. Immunohistochemistry study of clinical samples revealed that airway epithelial cells expressed both of these proteins. Cultured primary human airway epithelial cells expressed MMP-1, and MMP-1 was further induced by stimulation with EREG or heat-killed SA (HKSA). The induction of MMP-1 by HKSA was blocked by an antibody against EREG, suggesting that endogenous EREG induces MMP-1 after stimulation with HKSA. EREG and MMP-1 were found to be elevated in nasal polyp and uncinate tissues in patients with CRSwNP. Elevated expression of EREG and MMP-1 may be related to polyp formation in CRS, and colonization of SA might further enhance this process.

Background: Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known., Objectives: After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS., Methods: Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA., Results: C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P < .05) and correlated with levels of C5a (P < .01), local immunoglobulins (especially IgM, P < .0001), and anti-double-stranded DNA IgG (P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P < .0001)., Conclusion: Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium., Objective: We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease., Methods: Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase., Results: OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3-5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45 + cells were OSM + , and of the OSM + cells, 56% ± 7% were CD16 + Siglec-8 - , indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45 + events from matched blood samples (n = 5) were OSM + , suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM + neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM + neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05)., Conclusions: Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP., Objective: We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms., Methods: Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging., Results: Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P < .05). IL-13 stimulation of HNECs and BEAS-2B cells dominantly induced eotaxin-3 expression, which was significantly inhibited by PPIs (P < .05). Patients with CRS taking PPIs also showed lower in vivo eotaxin-3 levels compared with those without PPIs (P < .05). Using intracellular pH imaging and altering extracellular K + , we found that IL-13 enhanced H + ,K + -exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay., Conclusion: Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Chronic rhinosinusitis (CRS) is likely a biologically heterogeneous disease process. Current guidelines propose subclassification using polyp status while others propose using mucosal eosinophilia. We hypothesized that appropriate CRS subclassification would increase homogeneity of baseline symptoms, and identify characteristic symptoms of each subtype., Methods: A total of 57 CRS patients undergoing surgery prospectively completed a preoperative battery of 73 questions relating to symptoms including the 22-item Sino-Nasal Outcome Test (SNOT-22) and 29-item Patient-Reported Outcomes Measurement Information System (PROMIS-29) general quality of life (QOL) measures. Eosinophilic cationic protein (ECP) levels were determined from ethmoid, uncinate, and polyp tissue homogenates using enzyme-linked immunosorbent assay (ELISA) and normalized to total protein. Patients were classified as eosinophilic (eCRS) or non-eosinophilic (neCRS) using a 95th percentile threshold established from control tissue from 82 patients without CRS. Separate pairwise comparisons were performed on patient-reported symptoms using polyp and eosinophilic status., Results: Of the 57 patients, 28 had CRS with nasal polyps (CRSwNP); 27 of 57 patients had eCRS (CRSwNP, n = 21; CRS without nasal polyps [CRSsNP], n = 6). CRSwNP patients had increased need to blow nose, frequency of nasal congestion, more severe difficulty breathing through nose, more severe nasal discharge, but less cough (p < 0.05). eCRS patients had more bothersome loss of taste/smell, ear pain, sneezing, severe difficulty breathing through nose, and severe nasal congestion compared to neCRS patients (p < 0.05)., Conclusion: Subclassifying CRS with symptoms alone is difficult with neither polyp status nor eosinophilia giving a distinctive clinical symptom profile. However, certain symptoms may help otolaryngologists identify CRS subtypes, which may help guide future treatments. Further validation and evaluation of prognosis following treatment is required to evaluate appropriate means of subclassifying CRS., (© 2015 ARS-AAOA, LLC.)

Aspergillus fumigatus (AF) infection and sensitization are common and promote Th2 disease in individuals with asthma. Innate immune responses of bronchial epithelial cells are now known to play a key role in determination of T cell responses upon encounter with inhaled pathogens. We have recently shown that extracts of AF suppress JAK-STAT signaling in epithelial cells and thus may promote Th2 bias. To elucidate the impact of AF on human bronchial epithelial cells, we tested the hypothesis that AF can modulate the response of airway epithelial cells to favor a Th2 response and explored the molecular mechanism of the effect. Primary normal human bronchial epithelial (NHBE) cells were treated with AF extract or fractionated AF extract before stimulation with poly I:C or infection with human rhinovirus serotype 16 (HRV16). Expression of CXCL10 mRNA (real-time RT-PCR) and protein (ELISA) were measured as markers of IFN-mediated epithelial Th1-biased responses. Western blot was performed to evaluate expression of IFN regulatory factor-3 (IRF-3), NF-κB, and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11), which are other markers of Th1 skewing. Knockdown experiments for protease-activated receptor-2 (PAR-2) and PTPN11 were performed to analyze the role of PAR-2 in the mechanism of suppression by AF. AF and a high-molecular-weight fraction of AF extract (HMW-AF; > 50 kD) profoundly suppressed poly I:C- and HRV16-induced expression of both CXCL10 mRNA and protein from NHBE cells via a mechanism that relied upon PAR-2 activation. Both AF extract and a specific PAR-2 activator (AC-55541) suppressed the poly I:C activation of phospho-IRF-3 without affecting activation of NF-κB. Furthermore, HMW-AF extract enhanced the expression of PTPN11, a phosphatase known to inhibit IFN signaling, and concurrently suppressed poly I:C-induced expression of both CXCL10 mRNA and protein from NHBE cells. These results show that exposure of bronchial epithelial cells to AF extract suppressed poly I:C and HRV16 signaling via a mechanism shown to involve activation of PAR-2 and PTPN11. This action of AF may promote viral disease exacerbations and may skew epithelial cells to promote Th2 inflammation in allergic airway disorders mediated or exacerbated by AF, such as asthma and chronic rhinosinusitis.

Background: Epithelial barrier dysfunction is thought to play a role in many mucosal diseases, including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis., Objective: The objective of this study was to investigate the role of oncostatin M (OSM) in epithelial barrier dysfunction in human mucosal disease., Methods: OSM expression was measured in tissue extracts, nasal secretions, and bronchoalveolar lavage fluid. The effects of OSM stimulation on barrier function of normal human bronchial epithelial cells and nasal epithelial cells cultured at the air-liquid interface were assessed by using transepithelial electrical resistance and fluorescein isothiocyanate-dextran flux. Dual-color immunofluorescence was used to evaluate the integrity of tight junction structures in cultured epithelial cells., Results: Analysis of samples from patients with CRS showed that OSM mRNA and protein levels were highly increased in nasal polyps compared with those seen in control uncinate tissue (P < .05). OSM levels were also increased in bronchoalveolar lavage fluid of allergic asthmatic patients after segmental allergen challenge and in esophageal biopsy specimens from patients with eosinophilic esophagitis. OSM stimulation of air-liquid interface cultures resulted in reduced barrier function, as measured by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran flux (P < .05). Alterations in barrier function by OSM were reversible, and the viability of epithelial cells was unaffected. OSM levels in lysates of nasal polyps and uncinate tissue positively correlated with levels of α2-macroglobulin, a marker of epithelial leak, in localized nasal secretions (r = 0.4855, P < .05)., Conclusions: These results suggest that OSM might play a role in epithelial barrier dysfunction in patients with CRS and other mucosal diseases., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti-TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2-dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti-TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α-dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis.

We announce here the complete genome sequence of the Pseudomonas aeruginosa mucoid strain FRD1, isolated from the sputum of a cystic fibrosis patient. The complete genome of P. aeruginosa FRD1 is 6,712,339 bp. This genome will allow comparative genomics to be used to identify genes associated with virulence, especially those involved in chronic pulmonary infections., (Copyright © 2015 Silo-Suh et al.)

Background: Although chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by Th2 inflammation, the mechanism underlying the onset and amplification of this inflammation has not been fully elucidated. Dendritic cells (DCs) are major antigen-presenting cells, central inducers of adaptive immunity and critical regulators of many inflammatory diseases. However, the presence of DCs in CRS, especially in nasal polyps (NPs), has not been extensively studied., Objective: The objective of this study was to characterize DC subsets in CRS., Methods: We used real-time PCR to assess the expression of mRNA for markers of myeloid DCs (mDCs; CD1c), plasmacytoid DCs (pDCs; CD303) and Langerhans cells (LCs; CD1a, CD207) in uncinate tissue (UT) from controls and patients with CRS as well as in NP. We assayed the presence of DCs by immunohistochemistry and flow cytometry., Results: Compared to UT from control subjects (n = 15) and patients with CRS without NP (CRSsNP) (n = 16) and CRSwNP (n = 17), mRNAs for CD1a and CD1c were significantly elevated in NPs (n = 29). In contrast, CD207 mRNA was not elevated in NPs. Immunohistochemistry showed that CD1c(+) cells but not CD303(+) cells were significantly elevated in NPs compared to control subjects or patients with CRSsNP. Flow cytometric analysis showed that CD1a(+) cells in NPs might be a subset of mDC1s and that CD45(+) CD19(-) CD1c(+) CD11c(+) CD141(-) CD303(-) HLA-DR(+) mDC1s and CD45(+) CD19(-) CD11c(+) CD1c(-) CD141(high) HLA-DR(+) mDC2s were significantly elevated in NPs compared to UT from controls and CRSsNP, but CD45(+) CD11c(-) CD303(+) HLA-DR(+) pDCs were only elevated in NPs compared to control UT., Conclusion and Clinical Relevance: Myeloid DCs are elevated in CRSwNP, especially in NPs. Myeloid DCs thus may indirectly contribute to the inflammation observed in CRSwNP., (© 2014 John Wiley & Sons Ltd.)

Background: Profound edema or formation of a pseudocyst containing plasma proteins is a prominent characteristic of nasal polyps (NP). However, the mechanisms underlying NP retention of plasma proteins in the submucosa remain unclear. Recently, we reported that impairment of fibrinolysis causes excessive fibrin deposition in NP and this might be involved in the retention of plasma proteins. Although the coagulation cascade plays a critical role in fibrin clot formation at extravascular sites, the expression and role of coagulation factors in NP remain unclear., Objective: The objective of this study was to investigate the expression of coagulation factors in patients with chronic rhinosinusitis (CRS)., Methods: Sinonasal tissues were collected from patients with CRS and control subjects. We assayed mRNA for factor XIII-A (FXIII-A) by using real-time PCR and measured FXIII-A protein by means of ELISA, immunohistochemistry, and immunofluorescence., Results: FXIII-A mRNA levels were significantly increased in NP tissue from patients with CRS with NP (P < .001) compared with uncinate tissue from patients with CRS or control subjects. Similarly, FXIII-A protein levels were increased in NP. Immunofluorescence analysis revealed that FXIII-A expression in inflammatory cells and FXIII-A(+) cell numbers were significantly increased in NP. Most FXIII-A staining was observed within CD68(+)/CD163(+) M2 macrophages in NP. Levels of FXIII-A correlated with markers of M2 macrophages, suggesting that M2 macrophages are major FXIIIA-producing cells in NP., Conclusion: Overproduction of FXIII-A by M2 macrophages might contribute to the excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with NP., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with TH2-dominant inflammation. Thymic stromal lymphopoietin (TSLP) is a cytokine that triggers dendritic cell-mediated TH2 inflammatory responses and that enhances IL-1-dependent TH2 cytokine production in mast cells. Although increased TSLP mRNA levels have been found in nasal polyps (NPs), expression of TSLP protein and its function in patients with chronic rhinosinusitis (CRS) have not been fully explored., Objectives: The objective of this study was to investigate the role of TSLP in patients with CRS., Methods: We investigated the presence and stability of TSLP protein in NPs using ELISA and Western blotting and investigated the function of TSLP in nasal tissue extracts with a bioassay based on activation of human mast cells., Results: Although TSLP mRNA levels were significantly increased in NP tissue from patients with CRSwNP compared with uncinate tissue from patients with CRS or control subjects, TSLP protein was significantly decreased in NP tissue, as detected by using the commercial ELISA kit. We found that recombinant TSLP was time-dependently degraded by NP extracts, and this degradation was completely inhibited by a protease inhibitor cocktail, suggesting that TSLP is sensitive to tissue proteases. Interestingly, NP extract-treated TSLP had higher activity in mast cells, although the amount of full-length TSLP was reduced up to 85%. NP extracts significantly enhanced IL-1β-dependent IL-5 production in mast cells compared with uncinate tissue homogenates, and responses were significantly inhibited by anti-TSLP, suggesting that NPs contain biologically relevant levels of TSLP activity., Conclusion: TSLP and its metabolic products might play an important role in the inflammation seen in patients with CRSwNP., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Rationale: Nasal polyps (NPs) are characterized by intense edema or formation of pseudocysts filled with plasma proteins, mainly albumin. However, the mechanisms underlying NP retention of plasma proteins in their submucosa remain unclear., Objectives: We hypothesized that formation of a fibrin mesh retains plasma proteins in NPs. We assessed the fibrin deposition and expression of the components of the fibrinolytic system in patients with chronic rhinosinusitis (CRS)., Methods: We assessed fibrin deposition in nasal tissue from patients with CRS and control subjects by means of immunofluorescence. Fibrinolytic components, d-dimer, and plasminogen activators were measured using ELISA, real-time PCR, and immunohistochemistry. We also performed gene expression and protein quantification analysis in cultured airway epithelial cells., Measurements and Main Results: Immunofluorescence data showed profound fibrin deposition in NP compared with uncinate tissue (UT) from patients with CRS and control subjects. Levels of the cross-linked fibrin cleavage product protein, d-dimer, were significantly decreased in NP compared with UT from patients with CRS and control subjects, suggesting reduced fibrinolysis (P < 0.05). Expression levels of tissue plasminogen activator (t-PA) mRNA and protein were significantly decreased in NP compared with UT from patients with CRS and control subjects (P < 0.01). Immunohistochemistry demonstrated clear reduction of t-PA in NP, primarily in the epithelium and glands. Th2 cytokine-stimulated cultured airway epithelial cells showed down-regulation of t-PA, suggesting a potential Th2 mechanism in NP., Conclusions: A Th2-mediated reduction of t-PA might lead to excessive fibrin deposition in the submucosa of NP, which might contribute to the tissue remodeling and pathogenesis of CRS with nasal polyps.

Background: Although chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) is characterized by T(H)2 inflammation, the role of mast cells is poorly understood., Objective: The objective of this study was to investigate the presence, localization, and phenotype of mast cells in patients with CRS., Methods: We collected nasal tissue and nasal lavage fluid from patients with CRS and control subjects. We analyzed mRNA for the mast cell proteases tryptase, chymase, and carboxypeptidase A3 by using real-time PCR and measured mast cell protease proteins by using ELISA, immunohistochemistry, and immunofluorescence., Results: Tryptase mRNA was significantly increased in nasal polyps (NPs) from patients with CRSwNP (P< .001) compared with uncinate tissue from patients with CRS or control subjects. Tryptase protein was also elevated in NPs and in nasal lavage fluids from patients with CRSwNP. Immnohistochemistry showed increased numbers of mast cells in epithelium and glands but not within the lamina propria in NPs. The mast cells detected in the epithelium in NPs were characterized by the expression of tryptase and carboxypeptidase A3 but not chymase. Mast cells expressing all the 3 proteases were abundant within the glandular epithelium of NPs but were not found in normal glandular structures., Conclusions: Herein we demonstrated a unique localization of mast cells within the glandular epithelium of NPs and showed that mast cells in NPs have distinct phenotypes that vary by tissue location. Glandular mast cells and the diverse subsets of mast cells detected may contribute to the pathogenesis of CRSwNP., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with T(H)2-dominant inflammation, including eosinophilia, which is in contrast to chronic rhinosinusitis (CRS) without nasal polyps (NPs). CC chemokine ligand 18 (CCL18)/pulmonary and activation-regulated chemokine is known to recruit naive T cells, B cells, and immature dendritic cells, as well as to activate fibroblasts. CCL18 is thought to be involved in T(H)2-related inflammatory diseases, including asthma and atopic dermatitis., Objective: The objective of this study was to investigate the expression of CCL18 in patients with CRS., Methods: Using NP tissue and uncinate tissue (UT) from control subjects and patients with CRS, we examined the expression of CCL18 mRNA using real-time PCR and measured CCL18 protein using ELISA, Western blotting, and immunofluorescence., Results: Compared with UT tissue from control subjects, CCL18 mRNA levels were significantly increased in NPs (P < .001) and UT (P < .05) from patients with CRSwNP but not in UT from patients with CRS without NPs. Similarly, CCL18 protein levels were increased in NPs and UT from patients with CRSwNP, and levels were even higher in patients with Samter's triad. Immunohistochemical analysis revealed CCL18 expression in inflammatory cells, and CCL18(+) cell numbers were significantly increased in NPs. Immunofluorescence data showed colocalization of CCL18 in CD68(+)/CD163(+)/macrophage mannose receptor-positive M2 macrophages and tryptase-positive mast cells in NPs. Levels of CCL18 correlated with markers of M2 macrophages but not with tryptase levels, suggesting that M2 macrophages are major CCL18-producing cells in NPs., Conclusion: Overproduction of CCL18 might contribute to the pathogenesis of CRSwNP through its known activities, which include recruitment of lymphocytes and dendritic cells, activation of fibroblasts, and initiation of local inflammation., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Chronic rhinosinusitis (CRS) is a heterogeneous chronic disease characterized by local inflammation of the sinonasal tissues. The pathogenesis of CRS remains controversial, but it has been associated with the accumulation of various immune and inflammatory cells in sinus tissue., Objectives: The objective of this study was to investigate the expression of the chemokine CCL23, which is known to bind to CCR1 and recruit monocytes, macrophages, and dendritic cells, in patients with CRS., Methods: We collected nasal tissue from patients with CRS and control subjects. We assayed mRNA for CCL23 by using real-time PCR and measured CCL23 protein by means of ELISA, immunohistochemistry, and immunofluorescence., Results: CCL23 mRNA levels were significantly increased in nasal polyps (NPs) from patients with CRS with nasal polyps (CRSwNP; P < .05) compared with inferior turbinate and uncinate tissue from patients with CRS or control subjects. CCL23 protein levels were also increased in NPs, although these levels were not statistically significant. Immunohistochemical analysis revealed CCL23 expression in mucosal epithelial cells and inflammatory cells, but accumulation of CCL23(+) inflammatory cells occurred only in NPs. Immunofluorescence data showed CCL23 colocalization with eosinophil cationic protein-positive eosinophils. The concentration of CCL23 in NPs positively correlated with the concentration of eosinophil cationic protein, suggesting that eosinophils are major CCL23-producing cells in NPs. Finally, we found that CCL23 protein levels were significantly increased in NPs from patients with CRSwNP with aspirin sensitivity., Conclusion: Overproduction of CCL23 in NPs might contribute to the pathogenesis of eosinophilic CRSwNP through the recruitment of CCR1(+) inflammatory cells, including monocytes and macrophages, and the amplification of local inflammation., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: B-cell responses may play a role in the pathogenesis of nasal polyposis via local IgA and IgE production and activation of eosinophils and mast cells. B-cell attracting chemokines may therefore have relevance in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNPs) Methods: Polyp and inferior turbinate tissues were obtained from CRSwNPs, CRS without NPs (CRSsNPs), and control patients; ELISA and reverse-transcription polymerase chain reaction were used to detect levels of protein and mRNA for selected B-cell chemokines (B-cell attracting chemokine 1 [CXCL13/BCA-1/BLC]), thymus expressed chemokine (CCL25/TECK), mucosae-associated epithelial chemokine (CCL28/MEC), stromal cell-derived factor-1alpha (CXCL12/SDF-1alpha), and selected chemokine receptor genes (CXCR4, CXCR5, and CXCR7)., Results: BCA-1 and SDF-1alpha protein levels were significantly increased in polyp tissue compared with turbinate tissue from CRSsNP patients and controls (p < 0.05 and p < 0.01, respectively). Differences in TECK and MEC were not significant. For mRNA, expression of BCA-1 was significantly up-regulated in polyp tissue and levels correlated with CD20 mRNA expression. Additionally, significant up-regulation of mRNA for the SDF-1alpha receptors CXCR7 and CXCR4 was detected in polyps, while there was a trend for up-regulation of the BCA-1 receptor CXCR5., Conclusion: Elevated levels of the BCA-1 and SDF-1alpha and their receptors may account for an increased presence of B cells and their products, contributing to eosinophilic inflammation in patients with CRSwNP.

In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF(ts1). The mutations that resulted in a temperature-sensitive phenotype in mSF(ts1) were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 degrees C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF(ts1) was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF(ts1) for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.

Chronic lung infections caused by Pseudomonas aeruginosa are the leading cause of morbidity and mortality for cystic fibrosis (CF) patients. Adaptation of P. aeruginosa to the CF lung results in the loss of acute virulence determinants and appears to activate chronic virulence strategies in this pathogen. In order to identify such strategies, a random transposon mutagenesis was performed and 18 genes that were required for optimal infection of alfalfa seedlings by FRD1, a CF isolate of P. aeruginosa, were recognized. The largest subset of genes (seven of the 18), were associated with central carbon metabolism, including the gene that encodes isocitrate lyase (ICL), aceA. Because FRD1 is avirulent in animal infection models, we constructed an ICL mutant in P. aeruginosa strain PAO1 in order to assess the requirement of ICL in mammalian infection. The PAO1 ICL mutant was less virulent in the rat lung infection model, indicating that ICL is required for the pathogenesis of P. aeruginosa in mammals. Furthermore, FRD1 showed increased ICL activity and expression of an aceA : : lacZ fusion compared to PAO1. We suggest that upregulation of ICL occurred during adaptation of FRD1 to the CF lung and that some of the novel virulence mechanisms employed by FRD1 to infect alfalfa seedlings may be the same mechanisms P. aeruginosa relies upon to persist within human niches.

Background: Recent studies have suggested that Staphylococcus aureus secrete superantigenic toxins that play a role in the etiology of chronic rhinosinusitis with nasal polyposis (CRSwNP). Twenty S. aureus superantigens (SAg's) have been identified, each of which bind the V beta-region of the T-cell receptor (TCR) outside the peptide-binding site. Approximately 50 distinct V beta-domains exist in the human repertoire, and distinct SAg's will bind only particular domains generating a pattern of V beta-enrichment in lymphocytes dependent on the binding characteristics of a given toxin. The aim of this study was to analyze the pattern of V beta-expression in polyp-derived lymphocytes from CRSwNP patients., Methods: Polyps were harvested from 20 patients with CRSwNP and 3 patients with antrochoanal polyps. Flow cytometry was used to analyze the V beta-repertoire of polyp-derived CD4+ and CD8+ lymphocytes. Data were analyzed in light of the known skewing associated with SAg exposure in vivo and in vitro. Skewing was defined as a percentage of V beta-expression >2 SD of that seen in normal blood., Results: Seven of 20 subjects exhibited skewing in V beta-domains with strong associations with S. aureus SAg's. The three antrochoanal polyps failed to show any significant V beta-skewing., Conclusion: This study establishes evidence of S. aureus SAg-T-cell interactions in polyp lymphocytes of 35% of CRSwNP patients. Although these results are consistent with intranasal exposure of polyp lymphocytes to SAg's, additional study is necessary to establish the role of these toxins in disease pathogenesis.

Background: The superantigen (SAg) hypothesis of chronic rhinosinusitis (CRS) suggests that toxins within the nose stimulate massive oligoclonal expansion of T-cell populations with subsequent eosinophil recruitment and tissue inflammation. SAgs are capable of activating 1 x 10(4) more lymphocytes than conventional antigens by binding specific Vbeta-domains of the T-cell receptor (TCR). The net effect is skewing from the normal Vbeta-profile by oligoclonal expansion of specific domains. This study will assess polyp tissue for evidence of an SAg response in CRS with nasal polyps (CRSwNP)., Methods: This study consists of a prospective analysis of 18 CRSwNP subjects undergoing sinus surgery. Flow cytometry was used to analyze the distribution of 24 specific TCR V beta-domains in lymphocytes from polyp tissue and blood. Evidence of oligoclonal expansion was tabulated for each specimen and defined as mean normative percentage + 2 SD., Results: Eighteen of 18 CRSwNP subjects showed expansion of polyp lymphocytes expressing TCRs with specific V beta-domains. The average number of V beta-clones per CRSwNP subject was seven in polyp lymphocytes but only two in blood lymphocytes., Conclusion: The current results indicate that polyp lymphocytes exhibit significant oligoclonal expansion of specific V beta-domains. These data are considered diagnostic of a SAg effect. The corresponding blood profile is much less, suggesting that the nose is the primary source of stimulus. Although the trigger(s) for this expansion are unknown, these data are consistent with the hypothesis that staphylococcal toxins are central to the development of CRSwNP.

To facilitate study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, several genetic tools were developed. These tools include a series of cassettes carrying (a) the minimal sequence for the origin of transfer (oriT) of RP4 plasmid for introducing plasmid into P. aeruginosa via conjugation, (b) a minimal sequence for P. aeruginosa replicon (stabilizing fragment or SF) for maintenance of plasmids in P. aeruginosa, and (c) the transcriptionally non-polar tetracycline resistance gene (TcR) for insertional mutagenesis. Additional genetic constructs include (d) two conjugative and suicide lacZ reporter fusion plasmids for studying gene expression at the transcriptional or translational level, (e) a gentamicin resistant promoter-probing mini-Tn5 lacZ, and (f) a tightly regulated T7 promoter/repressor system to control gene expression in P. aeruginosa.

The manufacturing of PrePen (benzylpenicilloyl-polylysine) was suspended from September 2000 through November 2001 because of Food and Drug Administration intervention. PrePen is penicilloyl bound to polylysine and is considered to be the major determinant of penicillin metabolism; it is used as a skin-testing reagent to detect immunoglobulin E antibodies in people with a history of penicillin allergy. Because of the uncertainty of whether PrePen would be made available in the future, we attempted to make benzylpenicilloyl-polylysine in our laboratory using a modification of a previously described method. Subsequently, we compared our preparation with PrePen by enzyme-linked immunosorbent assay and skin testing. Using a patient previously documented to have a positive reaction to PrePen by history and intradermal skin testing, a puncture test to PrePen as well as dilutions of benzylpenicilloyl-polylysine was negative. Positive intradermal tests were performed on PrePen and benzylpenicilloyl-polylysine at 5 mg/mL. The enzyme-linked immunosorbent assay was performed to compare the antigenicity of PrePen and benzylpenicilloyl-polylysine and revealed similar binding to both. Subsequently, cutaneous testing on 10 additional penicillin-allergic people was performed, with corroborative results. Our benzylpenicilloyl-polylysine production methodology could prove useful to those who do not have access to PrePen or another similar reagent.

Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of dsbA in the mucoid PDO300 background did not affect alginate production. Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production. This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.

Zwittermicin A is a novel antibiotic produced by Bacillus cereus UW85, which suppresses certain plant diseases in the laboratory and in the field. We developed a rapid method for large-scale purification of zwittermicin A and then studied the in vitro activity of zwittermicin A against bacteria, fungi, and protists. Zwittermicin A was highly active against the Oomycetes and their relatives, the algal protists, and had moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi. Zwittermicin A was more active against bacteria and fungi at pH 7-8 than at pH 5-6. When zwittermicin A was combined with kanosamine, another antibiotic produced by B. cereus, the two acted synergistically against Escherichia coli and additively against Phytophthora medicaginis, an Oomycete. The results indicate that there are diverse potential applications of this new class of antibiotic.

Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis. We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P. medicaginis. We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide. Both antibiotics prevented disease of alfalfa seedlings caused by P. medicaginis. Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P. medicaginis, and antibiotic B caused swelling of the germ tubes. Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa. Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity. Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain. The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression. Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression. These results demonstrate that B. cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.